Elucidation of the functional elements of the malic enzyme promoter has continued with a series of experiments designed to determine the cis-acting regulatory elements necessary for basal transcription of this housekeeping gene. Using a cell-free in vitro transcription system of HeLa cell extracts, we found that maximal transcriptional activity from the malic enzyme promoter was imparted by a 5' flanking region containing 881 base pairs proximal to the major transcription initiation site. Along with the aforementioned studies, we are also examining thyroid hormone regulation of transcription of the malic enzyme gene by studying the effect of thyroid hormone treatment on the binding of trans-acting factors to the malic enzyme promoter. Using gel mobility shift assays, we found the treatment of hypothyroid rats with high doses of triiodothyronine resulted in a greater than 2-fold increase in the binding of specific hepatic protein(s) to the unique 10 base pair repeat at -71. This binding activity was present in other rat tissues as well as HeLa and H35 cells but only in liver was the activity regulated by thyroid hormone indicating that the mechanism of thyroid hormone stimulation of malic enzyme gene expression in liver probably involves a tissue-specific up-regulation of specific transacting transcription factor(s). Current studies include identification, isolation, and cloning of the thyroid hormone-regulated transcription factors.